A micro-LED array based platform for spatio-temporal optogenetic control of various cardiac models

Optogenetics relies on dynamic spatial and temporal control of light to address emerging fundamental and therapeutic questions in cardiac research. In this work, a compact micro-LED array, consisting of 16 × 16 pixels, is incorporated in a widefield fluorescence microscope for controlled light stimulation. We describe the optical design of the system that allows the micro-LED array to fully cover the field of view regardless of the imaging objective used. Various multicellular cardiac models are used in the experiments such as channelrhodopsin-2 expressing aggregates of cardiomyocytes, termed cardiac bodies, and bioartificial cardiac tissues derived from human induced pluripotent stem cells. The pacing efficiencies of the cardiac bodies and bioartificial cardiac tissues were characterized as a function of illumination time, number of switched-on pixels and frequency of stimulation. To demonstrate dynamic stimulation, steering of calcium waves in HL-1 cell monolayer expressing channelrhodopsin-2 was performed by applying different configurations of patterned light. This work shows that micro-LED arrays are powerful light sources for optogenetic control of contraction and calcium waves in cardiac monolayers, multicellular bodies as well as three-dimensional artificial cardiac tissues.


Materials and methods
Three different cardiac models were used in our study.Multicellular aggregates of highly purified cardiomyocytes, termed cardiac bodies 39 , and bioartificial cardiac tissue (BCT), a composition of cardiomyocytes and fibroblasts derived from human pluripotent stem cells (hiPSCs) 40 .A transgenic HL-1 cell line 41 was used as a model for a 2D syncytium.All samples expressed the channelrhodopsin-2 variant H134R fused to eYFP.The hiPSCs were generated in house.The HL-1 cell line was kindly provided by Prof. Dr. Philipp Sasse.No animals were involved in this study.

Human induced pluripotent stem cell line culture
The human pluripotent stem cell line MHHi009-A-3 constitutively expresses the channelrhodopsin gene (ChR2, H134R variant5) in fusion to an eYFP reporter gene under the control of the ubiquitous CAG promoter (AAVS1-pCAG-ChR2-eYFP) and Zeocin resistance gene (ZeoR) under the control of the cardiac-specific α-myosin heavy chain (pMYH6) promoter.The hiPSC monolayers were cultivated in E8 medium 42 on Geltrex-coated flasks and passaged every 3-4 days using Accutase (both Life Technologies) 43 .Cells were kept at 37 °C and 5% CO 2 .

Cardiac differentiation
A schematic outline of the cardiac differentiation protocol is shown in Fig. 1a.HiPSC were differentiated into cardiomyocytes (CMs) using the established protocol based on the biphasic modulation of the WNT pathway 39 .Briefly, hiPSC monolayers were dissociated in single cells using Accutase (D3), then seeded at 0.2 × 10 6 cell/ml density in 125 ml Erlenmeyer flasks in E8 medium supplemented with 10 µM Rock Inhibitor (RI).Cells were kept in suspension culture on an orbital shaker (at 70 rpm at 37 °C and 5% CO2) for three days, allowing generation of 3D aggregates called embryoid bodies (EB).Differentiation was started with activation of the Wnt pathway using 5 µM CHIR99021 (Leibniz University, Hannover) for 24 h (D0), followed by the inhibition of Wnt pathway using 2 µM Wnt-C59 (Leibniz University, Hannover) for 72 h (D1-D3).Between D3 and D7, cells were cultivated in CDM-3 medium without any supplements and medium change was performed every other day.
Spontaneous contractions in the aggregates could be observed around D7. Fluorescence image of the cardiac bodies depicts the YFP signal from the ChR2 positive cells (Fig. 1b).Flow cytometry data shows high eYFP expression in transgenic hIPSC and differentiated cardiac cells MHHi009-A-3 compared to the mother cell lines MHHi009-A (Fig. 1c).At D8, cardiac selection was performed by using basic-serum-free medium (21969-DMEM, 1% Non-essential Amino Acids, 2 mM l-glutamine, 0.1 mM β-mercaptoethanol all from Life Technologies, 5.6 mg/l transferrin, 37.2 µg/l Sodium-selenite both from Sigma-Aldrich) supplemented with 100 µg/ml Zeomicin (Life Technologies) for 7 days.Efficiency of the cardiac differentiation and selection was analyzed via flow cytometry using typical cardiac markers, cardiac troponin T (cTnT) and myosin heavy chain (MYH) (96.7% and 98.9%, respectively) at day 15 (Fig. 1d).

Flow cytometry
Immunostaining and flow cytometry analysis on single cell suspension were performed as previously described 43 .Prior to antibody staining for intracellular markers, cells were fixed with ice-cold 90% methanol (15 min).Primary antibodies or the respective isotype controls, diluted in a PBS-based buffer (0.1% Triton-X 100, 0.5% BSA; both Sigma-Aldrich) were incubated with the cells for 1 h, followed by a 30 min incubation period with the appropriate secondary antibody (Donkey Anti-mouse IgG Alexa Fluor 647; 1:300, from Dianova, DE).All measurements were performed with the Accuri C6 (BD Biosciences) flow cytometer and data was analyzed using FlowJo_V10 (FlowJo LLC).Primary antibodies and their dilutions are listed in Table 1.
Isotype controls were used in the same concentration as the respective primary antibody for each staining.

Bioartificial cardiac tissue
Cardiac bodies were dissociated in single cell suspension using STEMdiff™ Cardiomyocyte Dissociation Kit (Stem Cell Technologies) according to manufacturer's instructions.Cardiomyocytes (1 × 10 6 cells per tissue) were then combined with irradiated foreskin fibroblasts (1 × 10 5 cells per tissue) in ratio of 10:1 into a hydrogel mixture composed of 0.9 mg/ml rat collagen type I (R&D System), 10% Geltrex™, and 2.5% 0.4 M NaOH (Sigma-Aldrich) 40 .The cell-matrix solution was poured into custom-made silicone molds with anchoring titanium rods at 6 mm distance and left for 30 min at 37 °C to allow solidification 44 .Bioartificial cardiac tissues (BCTs) were cultivated in BCT medium (DMEM F12 supplemented with 12% (v/v) horse serum (Life Technologies), 1 mM l-glutamine, 10 µg/ml insulin (Sigma-Aldrich), 1% penicillin-streptomycin (Life Technologies).Daily medium change was performed with fresh BCT medium supplemented with 30 µM l-ascorbic acid (Sigma-Aldrich).BCTs were cultivated for 7 days, then a progressive growing stretch was applied by stepwise distancing of the rods by 400 µm every 4 days until day 21 40,44 .Hydrogels, such as the mixture of Geltrex and Collagen I, naturally stimulate cells to form intercellular connections, which reduces the liquid content and thereby remodels the cardiac tissue 44 .This was observed for the BCTs used in this study, too, by monitoring the reorganization of the tissue using stereomicroscopy pictures over the 21 days of culture.A reduction in tissue diameter of approximately 40% was observed.Increased cellular interconnectivity was also demonstrated through the transition from sporadic contractions in the early days after production to a more synchronized and uniform contraction by day 21.
Figure 1e shows the fluorescence image of a bioartificial cardiac tissue with a uniform YFP expression on the entire tissue.
The sample preparation and staining were discussed in our previous study 18 .Briefly, 100,000 HL-1 cells expressing ChR2 were seeded in a 35 mm glass bottom dish (ibidi) coated with fibronectin (5 mg/l, Sigma-Aldrich) and gelatin (0.02%, Sigma-Aldrich) and incubated for further 24 to 48 h prior experiments.On the day of the experiments, cells were washed with Dulbecco phosphate buffer solution (DPBS, PanBiotech) without Ca 2+ and Mg 2+ and stained with 2 µl of 5 mM Cal-630 in 500 µl culture medium.After 45 min of incubation, the medium was replaced with 1 ml of fresh medium and cells were further incubated for 30-45 min before optogenetic experiments.

Experimental setup Imaging
A schematic diagram of the setup is shown in Fig. 2. The custom designed microscope consists of a pair of objectives, one in front of the camera (2×, 0.1 NA, Nikon, camera objective) and a second one directly above the sample (4×, 0.1 NA/10×, 0.25 NA/20×, 1 NA, Olympus, imaging objective).The objectives work with a corresponding tube lens (AC508-180-AB-ML, f = 180 mm, Thorlabs) arranged in a 4f configuration, as depicted in Fig. 2, to first magnify and then demagnify the image of the sample on the CMOS camera chip (Orca flash 4.0, Hamamatsu).The size of the field of view can be flexibly changed by using different imaging objectives.
Video microscopy of the contraction of the cardiac bodies and BCTs were performed in transillumination mode by an LED with a central wavelength of 590 nm (Solis-590C, Thorlabs).
For fluorescence imaging, an excitation filter (605/15, FF01-605/15-25, Semrock) was used to narrow the LED spectrum and an emission filter (655/40, FF02-655/40-25, Semrock) was integrated between the two tube lenses.The light source and filter set were chosen to match the excitation and emission spectrum of the fluorescent calcium dye, Cal-630.

Micro-LED array
Optical excitation of ChR2-expressing cells was realized using a Structured Micro Illumination Light Engine (SMILE, QubeDot) consisting of 256 squared pixels of micrometer-sized light emitting semiconductors arranged in an array of 16 × 16.Each single micro-LED is 50 × 50 µm 2 in size with a pixel pitch of 140 µm.SMILE was powered and controlled via a 5 V USB-cable connected to a computer.Each single pixel could be switched on and off independently by the provided software, which was also used to create looped animations.The emitted blue light (450 nm, central wavelength) was collected by an objective (20×, 0.5 NA, dry, Nikon) and a tube lens (f = 50 mm, Thorlabs) and implemented into the microscope by a dichroic mirror (T455 LPXR, Semrock) in between the 180 mm tube lenses, as depicted in Fig. 2.

Experimental methods
The emission power of the micro-LED array was measured via an optical power meter (PWM100USB, Thorlabs) either directly in front of the device to quantify the direct light output or at the sample plane to measure the power reaching the sample.
Periodic light excitation of the cardiac bodies was realized by creating animations consisting of single frames in the SMILE software.Depending on the desired illumination time and pacing frequency, one frame with active pixels was followed by a respective amount of empty frames.The refresh rate, which corresponds to the amount All videos were captured via a custom-made LabView program stored in tagged image file format (tiff) and binned to reduce the file size (for brightfield configuration) or to increase the signal-to-noise ratio (for fluorescence mode).Analysis of amplitude and duration of contraction was conducted by Myocyter 48 .The relatively large contraction amplitude rendered any motion artifacts neglectable, for example floating tissue residuals in the medium.In some samples, light from the micro-LEDs was visible in the camera, which can impede the image analysis.Therefore, a region of interest (ROI) was chosen to contain the outlines of the respective CB.
For optogenetic experiments, the cardiac bodies were placed in 35 mm dishes (ibidi) in complete medium and incubated at 37 °C and 5% CO 2 .After 1-3 days, they would attach to the bottom of the dish and were imaged using the 10× imaging objective.BCTs were incubated at 37 °C and 5% CO 2 .For imaging they were transferred to a 35 mm glass bottom dish with one of the titanium rods carefully removed to facilitate visible contraction.The BCTs were imaged with the 4× imaging objective.HL-1 cells expressing ChR2 were prepared and stained with Cal-630 dye as mentioned above.Calcium imaging was performed using the 20× imaging objective.
To acquire activation maps for steering of the calcium waves in 2D HL-1 syncytia, the Matlab-toolbox COSMAS 49 with a self-written GUI was used after applying a spatial Gaussian filter with 10 pixel radius in Fiji to the tiff images.For the wave steering experiments implemented in this work, calcium waves prior to and after the applied patterns were analyzed and plotted as activation maps.
All experiments on biological samples were carried out within limited time periods (less than 30 min) at room temperature (20 °C).

Characterization of imaging setup
In our system, the sample plane is imaged onto the CMOS chip by two compound microscopes, a magnifying one in the forward and a demagnifying one in the backward direction.Since the magnification is only valid for the focal length of the manufacturer's specified tube lens, the effective magnification of an objective (M Effective ) is given by objective, the size of the imaged micro-LED scales with the magnification of the microscope itself.Residual blue light from the sample plane, imaged onto the camera despite the dichroic mirror, always irradiates the same area at the CMOS chip as shown in Fig. 3b.
The sizes of a single pixel at the sample plane can be calculated as the product of the magnification of both imaging ensembles, which in theory are 62.5 µm, 25 µm or 12.5 µm for the three objectives, respectively.Figure 3c shows the experimentally measured pixel size and pitch as a function of camera binning.Due to the lower spatial resolution, the pixels and their pitch size can appear slightly bigger or smaller at the camera chip than the theoretical value (Fig. 3c pale lines) when using small magnification or higher binning.
Each single pixel in the micro-LED array provides at least 300 µW of output power, but does not scale linearly with increasing number of switched-on pixels.This is attributed to the common ground contact (cathode) and its resistance of the LED array.A higher amount of switched-on pixels leads to a higher current, which leads to a higher voltage loss at the common ground contact for all pixels and therefore reducing the current and optical output power of individual micro-LEDs.This behavior is called "multi pixel droop" 50 .A maximum power of 4.5 mW can be reached, when all pixels are switched on at the same time (Fig. 3d).The light reaching the sample plane is of significantly lower power: 120 µW, 90 µW or 149 µW maximum for the 4×, 10× and 20× imaging objective, respectively (Fig. 3e).The corresponding intensities at the sample plane are inversely related to the numbers of used pixels due to the multi pixel droop.Therefore, the maximum intensities of 1.7 mW/mm 2 , 8.5 mW/mm 2 and 52.3 mW/mm 2 for the 4×, 10× and 20× objectives, respectively, are achieved when only a single micro-LED is used.Precise control of the applied power at the sample plane can be achieved by pulse width modulation (PWM).It can be operated at a user-defined duty cycle with a minimum of 130 µs frame-time for the used device.For example, an animation consisting of one active and one inactive frame, looped in a frame rate yields an average power reduction of 50%.Here, PWM was tested and verified for 200 µs illumination time per frame.The power output is linearly proportional to the duty cycle (Fig. 3f).Table 2 summarizes the specifications of the setup using the different imaging objectives used.

Optical pacing of light-sensitive hiPSC cardiac bodies and bioartificial cardiac tissues
Cardiac bodies were examined using the 10× imaging objective in bright field mode (Fig. 4a).Illumination with blue light triggered contractions which were distinct from spontaneous contractions (Fig. 4b).Pacing the clusters was carried out at different pacing frequencies from 0.25 to 2 Hz using an illumination time of τ = 100 ms.We defined pacing efficiency as the relative number of successfully light-triggered contractions.While slower pacing frequencies yielded 100%, frequencies of 1 Hz and higher did not always elicit 1:1 contraction (Fig. 4c). Figure 4d shows examples of amplitudes for one cardiac body.While 0.5 Hz and 1 Hz pacing result in peaks clearly correlated to the excitatory stimulus, 2 Hz pacing led to irregular and unpredictable contractions.
We observe that most of the time, a single switched-on pixel with 5.3 µW at the sample plane is sufficient to successfully trigger a contraction at 0.5 Hz and τ = 100 ms.For shorter illumination time, τ = 33 ms and slightly higher pacing frequency, f = 0.6 Hz, the pacing efficiency decreased to 80% when illuminated with a single pixel, whereas six pixels, with total power of 21.7 µW at the sample plane were sufficient to reach a pacing efficiency of 100% (Fig. 4e, squares, n = 27).
For the BCTs which were ~ 1 mm in width and over 5 mm in length (Fig. 5a), the 4× imaging objective was used.BCTs were paced with 10 micro-LEDs at frequencies of 0.5 Hz, 1 Hz and 2 Hz (Fig. 5b).Three BCTs were investigated.One of the BCTs followed the pacing frequency of the micro-LED array (Fig. 5c squares) for all frequency tested, while one was successfully paced at 0.5 Hz and 1 Hz, but skipped every second excitation trigger at 2 Hz (Fig. 5c triangles).The contractions of the third BCT (Fig. 5c diamonds) were highly unpredictable, especially at 2 Hz pacing frequency (Fig. S2).Contraction frequencies were calculated as the inverse of the time interval between the two peak maxima.

Optical wave steering in a 2D cardiac syncytium
The micro-LED array provides a straightforward and intuitive interface to control each single emitter.Beside customized patterns of illumination, dynamic patterns are of crucial interest in cardiac optogenetic research, for example to change the chirality of native behavior of action potentials in ChR2-expressing cardiac monolayers 22 .In this section, we demonstrate dynamic light patterning using the micro-LED array to control calcium activity in 2D light-sensitive HL-1 cardiac monolayer.HL-1 cells are known to exhibit spontaneous spiral rotors or re-entries 51 .Three different dynamic light patterns for controlling and redirecting calcium waves were applied onto 2D syncytia of HL-1 cells: (1) a linear swipe (6 cycles at 0.6 Hz repetition rate, Fig. 6a), (2) a curved swipe rotated in a spiral around the center of the FOV (8 cycles at 0.5 Hz repetition rate, Fig. 6b), and (3) an inwarddirected spiral (5 cycles at 0.6 Hz repetition rate, Fig. 6c).Each dynamic pattern, or animation, was created in the SMILE software as a sequence of single static patterns.Since HL-1 cells exhibit random spontaneous waves which could vary from dish to dish, illumination patterns were applied sequentially at arbitrary time points during the experiment.Representative activation maps of calcium waves before and after application of micro-LEDs are shown in Fig. 6d-f, g-i, respectively.For all patterns, the calcium waves changed their direction and followed the general trajectory of the light pattern.The linear swipe was applied six times before the LEDs were switched off again.Figure 6d shows the calcium wave propagating from right to left of the field of view.Applying a linear, vertical train of blue light to the sample reversed the calcium wave direction from its initial leftwards flow direction towards right (Fig. 6g).Even though conduction was unimpeded prior to micro-LED application, the stimulated wave did not propagate through the upper half of the FOV, leading to a curved or spiral trajectory.
The curved pattern was applied and rotated eight times around the center of FOV before the LEDs were switched off again.Directly after light application, the initial leftwards directed flow (Fig. 6e) was changed and a spiral pattern of calcium wave could be observed (Fig. 6h).However, the spontaneous calcium wave immediately dominated after a single stimulated spiral trajectory.Hence, in this example, only a temporary modulation of calcium waves was implemented.www.nature.com/scientificreports/ The inward directed spiral light pattern led to a clear directed shift in calcium wave propagation.Instead of a spontaneous leftward flow (Fig. 6f), the stimulated calcium wave came from the top left and spread towards the bottom right (Fig. 6i), after the light pattern was applied five times.In contrast to the second pattern, this behavior was maintained for succeeding waves.
To quantify the success rates of the applied pattern, we rated a change of the initial directory of the calcium wave to be either successful (the wave followed the applied pattern), half successful (the calcium wave did not follow the applied pattern but drastically changed its direction), or not successful (the applied pattern had negligible influence on the calcium wave's direction).With this set of criteria, the linear swipe yields a success rate of 30% (n = 5), the curved rotating swipe yields 50% (n = 3), and the inwards directed spiral yields 33% (n = 3).These results are listed in Table 3 together with a depiction of the initial and resulting calcium wave's direction.

Discussion
We described a micro-LED platform for optogenetic actuation of ChR2-expressing cardiac models in vitro.The system is applicable for optogenetic control of a single cell, 2D monolayer up to whole tissue studies with the potential for high throughput data acquisition.The key component of our platform is the SMILE, a 16 × 16 array of 450 nm-emitting micro-LEDs which is imaged onto the respective sample.SMILEs can be produced with different emission wavelengths, with peak emission wavelengths ranging from 365 to 620 nm.Thus, the system is not only applicable to excite blue light sensitive ChR2, but could also be used to control other microbial opsins.For example, green emitting LED arrays could be used to inhibit cells expressing the outward proton pump, ArchT 52 .Whereas, red-emitting LED arrays would be useful for optogenetic stimulation of opsins which have optimal sensitivity at the orange to red wavelengths such as, Chrimson (λ ~ 590 nm 53 ), ChRmine (λ 500-600 nm 54 ) or ReaChR (λ ~ 590 nm and 630 nm 15 ).
The magnification and demagnification of the micro-LEDs scale with the magnification and the FOV of our microscope itself, ensuring the entire FOV can be covered by blue light without the need to change or adjust the illumination source.The total FOV and magnification of the microscope can be easily matched to the sample of interest.Furthermore, for each magnification we triggered and imaged contractions of an optogenetic cardiac model with a corresponding dimension scale, thus confirming the potential of this set-up.
A single 50 × 50 µm 2 -sized pixel from the SMILE directly emits 300 µW, corresponding to an intensity of 120 mW/mm 2 -two orders of magnitudes higher than the necessary intensity to excite ChR2 55 .Although, the irradiance at the sample is considerably lower, ~ 1.7 mW/mm 2 , 8.5 mW/mm 2 and 52.3 mW/mm 2 for the Table 3. Direction of calcium waves in transgenic HL-1 cells prior to and after three different dynamic patterns of blue light excitation.Green marked cells indicate calcium waves which followed the applied pattern, yellow ones were considered half successful and red ones non-successful.www.nature.com/scientificreports/4×, 10× and 20× objectives, respectively, these intensities are still sufficient to pace the cardiac samples.Optical power losses in the system could be further reduced by incorporating a collecting lens directly attached to the micro-LEDs.An advantage of using SMILE is that each single pixel can be individually addressed without any cross-talk between neighboring pixels 56 .This maximizes the flexibility of our system for simultaneous and dynamic excitation.
In addition to its flexibility, our platform is easily customizable to targeted sample size, making it also suitable for research at the scale of whole tissue slices.The emergence of hiPSC-CMs and their potential as a tool to model arrhythmogenic diseases have been vastly discussed [57][58][59] and non-invasive methods for control and analysis of such samples are demanded.We used multicellular cardiac bodies made of aggregated hiPSC-CMs as a model to show that our system is able to fulfill this demand.The intensity of a single switched-on pixel, 8.5 mW/mm 2 using 10× objective is sufficient to trigger contractions in our cardiac bodies with 97% success rate at 0.5 Hz and 100 ms illumination time.Slightly higher frequency and lower illumination time of 0.6 Hz and 33 ms yielded a success rate of 80% ± 12%.
Another scope of application, we explored in our work, lies in the rising interest of engineered cardiac patches.Despite progress in cardiac research, cardiac diseases are still the main factor for adult mortality and morbidity.Therefore, regeneration of cardiac function is still an important ongoing research field 60,61 .Cardiac tissue patches are currently being explored as candidates to aid repair and restore cardiac functionality.For example, hiPSC-CMs have been successfully used as implants in damaged hearts in animal models to improve cardiac function [62][63][64][65] .Recently, they have been proven to successfully integrate into the host myocardium of nonhuman primates 66 .However, electric coupling of the graft might be insufficient and thus a potential risk of arrhythmia, which is why modulation of the contraction frequency would be useful.The use of light-sensitive hiPSC-CMs and BCTs allows for thorough investigation in vitro and in vivo before considering clinical use of hiPSC-CMs.With our setup, we showed successful optical stimulation of cardiac patches, while parameters such as contraction frequency (Fig. 5c), amplitude (Fig. S1b), duration of contraction (Fig. S1c) and time to peak (Fig. S1d) and relaxation time (Fig. S1e), can be measured by all-optical means.These results display the potential of the setup for studies in cardiac patch therapy.
The micro-LED array can serve as an easy-to-use platform for high-throughput, contactless quantification of electrophysiological properties 67 .Such technology is of high interest in the context of cardiotoxicity screening for drug development 68 or therapeutic applications e.g.treatment of short QT syndrome 69 .Termination of ventricular tachycardia has been demonstrated in ChR2-transgenic mouse hearts 8 showing that termination success rate was highly dependent on the chosen illumination pattern.Feola et al. 70 demonstrated that specific patterns of blue light applied on a monolayer of ChR2-expressing neonatal rat cardiomyocytes could successfully block reentrant spirals and also used a predefined illumination pattern protocol to create the initial reentrant calcium wave.These studies highlight the need for customized stimulation patterns for treatment of arrhythmogenic conditions.
As a final application, we demonstrate that our system can steer and redirect propagating waves of cardiac action potential in a 2D syncytium model cell line.By switching on and off successive lines of the micro-LED array, we created a dynamic pattern of light that can overwrite an existing spontaneous wave.By creating a spiral animation, we redirected an initially plane wave into a spiral one.This can also be performed by applying precisely timed static illumination, for example in an S1-S2 protocol 38 .Our approach does not rely on precise timing of optogenetic activation, but on repeated application of the desired patterns.However, since the pattern of light is applied to a small region of interest within the sample, the resulting calcium wave is highly influenced by the interaction of the stimulated wave and the irregular propagating spontaneous calcium waves, as well as to conduction blocks existing in the HL-1 cell monolayer.This is shown in Fig. 6a, d, g wherein a linear swipe results in a curved calcium wave trajectory, and in the relative low success rate for steering in general, which did not exceed 50%.
For future studies, it would be interesting to investigate the influence of the used patterns on the success rate of optical wave steering.Contrary to the other two patterns, spirally rotating waves of cardiac action potential are known to be stable because they are self-sustaining 70,71 .To overcome the influence of calcium waves occurring outside the FOV, one can project the light patterns from the micro-LEDs on a larger region on the sample.In addition, several illumination parameters could be further optimized, such as the frequency, speed and timing of the applied dynamic light pattern.
Our experiments demonstrate the micro-LED array's high potential for investigating macroscopic waves in cardiac research.Implementation of the device has the potential to enhance and streamline current protocols for inducing and terminating spiral waves 72 .The ability to shape the excitation waves may be used for fast testing of anti-arrhythmic strategies, which might be even faster than computational models 73 .Thus, the simplicity of the micro-LED device is a promising approach for future investigations on optogenetic cardiac electrophysiology in vitro.

Limitations of the study
The presented study has certain limitations for in vitro cardiac research.Particularly, the current system has a limited illuminated region in a large cell culture area.Any spontaneous electrical propagation originating outside of this region would have a huge influence on the illuminated region's electrophysiology because they would interact with optogenetically steered electrical patterns.This scenario could be overcome by enlarging the illuminated FOV, which would require the use of a low magnification, high NA objective with significant light collection efficiency for fluorescence imaging.Furthermore, the presented optical system will benefit from an additional temperature control, which will be crucial for reliable and reproducible analysis.www.nature.com/scientificreports/At present, the micro-LED has a limited number of frames (2000) to build an animation.This constrains the use of PWM for pacing of cardiac models.In this case, an external shutter can be used to allow illumination with the desired frequency.

Conclusion
We presented a microscope platform for optogenetic cardiac research with a plug-and-play micro-LED array consisting of 16 × 16 directly-addressable blue-emitting micrometer-sized pixels.We showed the usefulness of our system as a flexible and easy-to-use tool for cardiac optogenetics to analyze contractile behavior of hiPSC-CMs and bioartificial cardiac tissue.10 micro-LEDs were shown to be sufficient for reliable cardiac pacing.Additionally, we illuminated dynamic light patterns using the inherent animation function of the SMILE to perturb and control the propagation of calcium waves in 2D syncytia of HL-1 cells.By careful design of our system, we ensured complete coverage of the FOV by the micro-LEDs independent of the chosen magnifying objective.The combination of high spatio-temporal resolution and parallel stimulation of the system can serve as an easy and powerful platform in cardiac optogenetic research.

Figure 1 .
Figure 1.(a) Overview of the cardiac differentiation of MHHi009-A-3 through biphasic modulation of WNT pathway, followed by antibiotic selection using Zeomicin resistance gene (ZeoR).(b) Differentiation at D15 resulted in cardiac bodies constitutively expressing eYFP.(c) Flow cytometry analysis showed homogenous expression of eYFP in MHHi009-A-3 hiPSC and CMs, compared to the not transgenic mother stem cell line MHHi009-A.(d) Cardiac selection was performed adding Zeomicin to the culture medium for 7 days, resulting in high expression of cardiac markers cardiac troponin T (cTnT) and myosin heavy chain (MYH) at D15. (e) Bioartificial cardiac tissues were generated, combining cardiomyocytes with irradiated fibroblasts and hydrogel solution.At D21, progressive growing stretch and spontaneous remodeling of the matrix resulted in compacted, homogenous, and beating tissues.Scale bar is 500 μm.
https://doi.org/10.1038/s41598-023-46149-1www.nature.com/scientificreports/ of time each frame was active, was dictated by the number of empty frames.Each animation was prepared in advance and stored as graphics interchange format (GIF).

Figure 2 .Figure 3 .
Figure 2. Imaging platform with integrated SMILE.Samples were illuminated from below and imaged from above by a 20×, 10×, or 4× objective with matching tube lens (f = 180 mm).Another pair of objective and tube lens demagnifies the image by 1.8 and projects it onto the CMOS chip.Blue light with a central wavelength of 450 nm emitted SMILE is captured by a 20× objective in combination with a tube lens (f = 50 mm) and guided onto the sample by a dichroic mirror (455 nm long pass).Tube lenses are adjusted in 4f-arrangement.

Figure 4 .Figure 5 .
Figure 4. Optogenetic excitation of cardiac bodies.(a) Image of a cardiac body stimulated with indicated pattern of micro-LEDs (blue dots, scale bar: 200 µm) and (b) a contraction trace of 5 paces at 0.5 Hz with a 10 s pause followed by other 5 illuminations.Light-triggered and spontaneous peaks in contraction are clearly visible.(c) Dependence of pacing efficiency of cardiac bodies on pacing frequency (0.25 Hz-2 Hz, n = 4, 10 pixels).Black line represents mean values ± standard deviation.(d) Representative contraction traces for pacing frequencies of 0.5 Hz, 1 Hz and 2 Hz with 100 ms illumination time and 30 µW (10 pixels).Contraction becomes irregular at 2 Hz.(e) Pacing efficiency as a function of number of active pixels for 0.5 Hz and 100 ms illumination time (circles, n = 24) and 0.6 Hz and 33 ms illumination time (squares, n = 27).Markers represent mean values ± standard deviation.The order of switched on pixels is given in the inset.

Figure 6 .
Figure 6.Representative activation maps of steered cardiac waves in HL-1 cells for three different animation patterns.(a-c) are the applied light patterns.(d-f) are the activation maps before applying the light patterns, (g-i) are the corresponding activation maps, respectively.For each map, the scale and timescale have been individually adjusted to better highlight the activation times of every pattern. https://doi.org/10.1038/s41598-023-46149-1 https://doi.org/10.1038/s41598-023-46149-1

Table 2 .
Specifications of micro-LED array and microscope, depending on the respective imaging objective.All the used objectives were manufactured by Evident (formerly Olympus).